Gamma-aminobutyric acid (GABA) can affect physiological processes in preimplantation embryos via GABAA and GABAB receptors.

Purpose: Several widely used substances (e.g., some therapeutics or food supplements) can act on gamma-aminobutyric acid (GABA) receptors, and we investigated whether the activation of these receptors could affect the preimplantation embryo. Methods: Transcripts of all GABA receptor subunits and selected proteins were examined using quantitative RT-PCR and immunohistochemistry. To analyze the effects of receptor activation, in vitro culture of mouse preimplantation embryos with natural and synthetic GABA receptor ligands was used. Results: We detected nine GABA receptor transcripts in mouse blastocysts and 14 GABA receptor transcripts in ovulated oocytes. The results of this study indicate that ionotropic GABAA receptors can be formed from α5, ß3, and γ3 (or δ, π) subunits, GABAA-ρ receptors can be formed from ρ2 subunits and metabotropic GABA receptors can be formed from GABAB1b and GABAB2 subunits in mouse blastocysts. Supplementing the culture medium with GABA at concentrations of 2-10 mM or with specific GABAA and GABAB receptor agonists (at concentrations of 10-100 µM) significantly increased the proportion of dead cells in blastocysts. The GABA-induced effects were prevented by pretreatment of embryos with GABAA and GABAB receptor antagonists. Conclusion: The results of this study indicate that GABA and synthetic GABA receptor ligands can negatively affect preimplantation embryos via GABAA and GABAB receptors.

Glutamate can act as a signaling molecule in mouse preimplantation embryos†.

Free amino acids are present in the natural environment of the preimplantation embryo, and their availability can influence early embryo development. Glutamic acid is one of the amino acids with the highest concentrations in female reproductive fluids, and we investigated whether glutamic acid/glutamate can affect preimplantation embryo development by acting through cell membrane receptors. Using reverse transcription-polymerase chain reaction, we detected 15 ionotropic glutamate receptor transcripts and 8 metabotropic glutamate receptor transcripts in mouse ovulated oocytes and/or in vivo developed blastocysts. Using immunohistochemistry, we detected the expression of two α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits, three kainate receptor subunits, and member 5 metabotropic glutamate receptor protein in blastocysts. Extracellular concentrations of glutamic acid starting at 5 mM impaired mouse blastocyst development, and this fact may be of great practical importance since glutamic acid and its salts (mainly monosodium glutamate) are widely used as food additives. Experiments with glutamate receptor agonists (in combination with gene expression analysis) revealed that specific AMPA receptors (formed from glutamate receptor, ionotropic, AMPA3 [GRIA3] and/or glutamate receptor, ionotropic, AMPA4 [GRIA4] subunits), kainate receptors (formed from glutamate receptor, ionotropic, kainate 3 [GRIK3] and glutamate receptor, ionotropic, kainate 4 [GRIK4] or glutamate receptor, ionotropic, kainate 5 [GRIK5] subunits), and member 5 metabotropic glutamate receptor (GRM5) were involved in this effect. The glutamic acid-induced effects were prevented or reduced by pretreatment of blastocysts with AMPA, kainate, and GRM5 receptor antagonists, further confirming the involvement of these receptor types. Our results show that glutamic acid can act as a signaling molecule in preimplantation embryos, exerting its effects through the activation of cell membrane receptors.

Canine Bone Marrow-derived Mesenchymal Stem Cells: Genomics, Proteomics and Functional Analyses of Paracrine Factors.

Adult stem cells have become prominent candidates for treating various diseases in veterinary practice. The main goal of our study was therefore to provide a comprehensive study of canine bone marrow-derived mesenchymal stem cells (BMMSC) and conditioned media, isolated from healthy adult dogs of different breeds. Under well-defined standardized isolation protocols, the multipotent differentiation and specific surface markers of BMMSC were supplemented with their gene expression, proteomic profile, and their biological function. The presented data confirm that canine BMMSC express important genes for differentiation toward osteo-, chondro-, and tendo-genic directions, but also genes associated with angiogenic, neurotrophic, and immunomodulatory properties. Furthermore, using proteome profiling, we identify for the first time the dynamic release of various bioactive molecules, such as transcription and translation factors and osteogenic, growth, angiogenic, and neurotrophic factors from canine BMMSC conditioned medium. Importantly, the relevant genes were linked to their proteins as detected in the conditioned medium and further associated with angiogenic activity in chorioallantoic membrane (CAM) assay. In this way, we show that the canine BMMSC release a variety of bioactive molecules, revealing a strong paracrine component that may possess therapeutic potential in various pathologies. However, extensive experimental or preclinical trials testing canine sources need to be performed in order to better understand their paracrine action, which may lead to novel therapeutic strategies in veterinary medicine.

Different response of embryos originating from control and obese mice to insulin in vitro.

The aim of the present work was to investigate the impact of maternal obesity on DNA methylation in ovulated oocytes, and to compare the response of in vitro-developing preimplantation embryos originating from control and obese mice to insulin. An intergenerational, diet-induced obesity model was used to produce outbred mice with an increased body weight and body fat. Two-cell and eight-cell embryos recovered from obese and control mice were cultured in a medium supplemented with 1 or 10 ng/ml insulin until blastocyst formation. In the derived blastocysts, cell proliferation, differentiation, and death rates were determined. The results of immunochemical visualization of 5-methylcytosine indicated a slightly higher DNA methylation in ovulated metaphase II oocytes recovered from obese females; however, the difference between groups did not reach statistical significance. Expanded blastocysts developed from embryos provided by control dams showed increased mean cell numbers (two and eight-cell embryos exposed to 10 ng/ml), an increased inner-cell-mass/trophectoderm ratio (two-cell embryos exposed to 1 ng/ml and eight-cell embryos exposed to 10 ng/ml), and a reduced level of apoptosis (two and eight-cell embryos exposed to 10 ng/ml). In contrast, embryos originating from obese mice were significantly less sensitive to insulin; indeed, no difference was recorded in any tested variable between the embryos exposed to insulin and those cultured in insulin-free medium. Real-time RT-PCR analysis showed a significant increase in the amount of insulin receptor transcripts in blastocysts recovered from obese dams. These results suggest that maternal obesity might modulate the mitogenic and antiapoptotic responses of preimplantation embryos to insulin.

Glucocorticoid receptor isoforms and effects of glucocorticoids in ovulated mouse oocytes and preimplantation embryos†.

To investigate possible involvement of glucocorticoid receptor (GR) in mediating effects of maternal stress or therapeutically administered glucocorticoids on early embryo, we analyzed the expression of GR subtypes in ovulated mouse oocytes and preimplantation embryos. RT-PCR analysis results showed that GRα and GRγ transcripts are relatively highly expressed in mouse oocytes, and both transcripts are present at lower amounts in preimplantation embryos. We also detected low expression of two other splice variants, GRß and a transcript orthologous to the human GR-P subtype, mainly at the blastocyst stage. Using western blot analysis, we detected several GR protein bands that differed in size between oocytes and preimplantation embryos. To compare the effects of corticosterone (a major endogenous glucocorticoid in rodents) and dexamethasone (a synthetic glucocorticoid) on early embryos, we cultured mouse preimplantation embryos in the presence of these glucocorticoids. Corticosterone showed a strong inhibitory effect on embryo development (starting from a 50 µM concentration), without a significant influence on apoptosis incidence. On the other hand, dexamethasone induced apoptosis in early embryo cells (starting from a 1.5 µM concentration), and its effect on embryo development was less detrimental than that found with the same dose of corticosterone. In summary, our results showed that different GR subtypes are expressed in ovulated mouse oocytes and preimplantation embryos and that the composition of GR subtypes changes during early embryo development. Moreover, we found significant differences in the effects of the two glucocorticoids on early embryo development, which might be associated with activation of different GR subtypes.

Overweight negatively affects outcome of superovulation treatment in female mice.

Superovulatory response is characterized by a high degree of variability and unpredictability. The aim of the present experimental study was to examine whether the amount of maternal body fat can influence the efficiency of ovarian stimulation with gonadotropins. Female mice of two body condition types, normal and obese, produced in a standardized two-generation model, were subjected to ovarian stimulation using eCG and hCG followed by natural mating. Produced ova and embryos were recovered on day 1 and day 4 of pregnancy respectively, and several quantitative, qualitative and developmental parameters were evaluated in them. The overall response of mouse females with normal and elevated amounts of body fat to superovulation was similar: They produced almost the same numbers of ova and embryos on average. Conversely, a higher number of immature oocytes, non-fertilized mature oocytes and lower-stage zygotes were collected from fat females. In both groups, the majority of fertilized oocytes was able to cleave and reach the higher stages of development. However, in the group of fat mice, a lower number of blastocysts was collected, and these blastocysts showed increased incidence of apoptotic cell death. In conclusion, although the response of normal and fat mice to superovulatory treatment was similar, the quality and developmental capacities of produced ova were lower in the group of fat donors.

The effect of maternal stress on blastocyst quality depends on maternal physiological status.

The effect of maternal stress on blastocyst quality, with respect to maternal metabolic status, was investigated in this study. We exposed female mice with different amounts of body fat to restraint stress and examined their blastocyst quality. Blood concentrations of corticosterone, leptin, adiponectin, insulin and glucose were measured in these females. Significantly lower stress-induced corticosterone elevations were observed in females with high and low amounts of body fat, indicating that the stress response was altered in these females. The basal leptin concentrations were significantly higher in females with high amounts of body fat than in females with low amounts of body fat, and stress induced different responses in these two groups of females. Our results showed that maternal stress can significantly increase the proportion of blastocysts that contain dead (apoptotic) cells in females with high and medium amounts of body fat. In females with low amounts of body fat, the proportion of blastocysts containing dead (apoptotic) cells was already increased before the stress exposure, and application of stress did not significantly change this parameter. Our results showed that the effects of maternal stress on early embryos can depend on the actual physiological status of the maternal organism exposed to stress.

Stress exposure during the preimplantation period affects blastocyst lineages and offspring development.

We found retardation of preimplantation embryo growth after exposure to maternal restraint stress during the preimplantation period in our previous study. In the present study, we evaluated the impact of preimplantation maternal restraint stress on the distribution of inner cell mass (ICM) and trophectoderm (TE) cells in mouse blastocysts, and its possible effect on physiological development of offspring. We exposed spontaneously ovulating female mice to restraint stress for 30 min three times a day during the preimplantation period, and this treatment caused a significant increase in blood serum corticosterone concentration. Microscopic evaluation of embryos showed that restraint stress significantly decreased cell counts per blastocyst. Comparing the effect of restraint stress on the two blastocyst cell lineages, we found that the reduction in TE cells was more substantial than the reduction in ICM cells, which resulted in an increased ICM/TE ratio in blastocysts isolated from stressed dams compared with controls. Restraint stress reduced the number of implantation sites in uteri, significantly delayed eye opening in delivered mice, and altered their behavior in terms of two parameters (scratching on the base of an open field test apparatus, time spent in central zone) as well. Moreover, prenatally stressed offspring had significantly lower body weights and in 5-week old females delivered from stressed dams, fat deposits were significantly lower. Our results indicate that exposure to stress during very early pregnancy can have a negative impact on embryonic development with consequences reaching into postnatal life.

Intestinal ischemia-reperfusion injury mediates expression of inflammatory cytokines in rats.

The small intestine is an organ with very well developed immunological activity, responsible for synthesis of specific inflammatory mediators that participate in causing the systemic inflammation that can occur after ischemia-reperfusion injury. The aim of our work was to study mRNA expression and protein concentration of inflammatory cytokines IL-10 and TNFα in the jejunal wall after intestinal ischemia-reperfusion injury (IRI). Cytokine concentration levels confirmed the direct effect of IRI on the inflammation process. The results refer to the changes in balance between pro-inflammatory and anti-inflammatory mediators and indicate that the predominant disturbance of homeostasis after intestinal IRI is present after 1 hour of reperfusion.

Do embryonic polar bodies commit suicide?

The extrusion and elimination of unnecessary gametic/embryonic material is one of the key events that determines the success of further development in all living organisms. Oocytes produce the first polar body to fulfill the maturation process just before ovulation, and release the second polar body immediately after fertilization. The aim of this study was to compile a physiological overview of elimination of polar bodies during early preimplantation development in mice. Our results show that three-quarters of the first polar bodies were lost even at the zygotic stage; the 4-cell stage embryos contained only one (second) polar body, and the elimination of second polar bodies proceeded continuously during later development. Both first and second polar bodies showed several typical features of apoptosis: phosphatidylserine redistribution (observed for the first time in the first polar body), specific DNA degradation, condensed nuclear morphology, and inability to exclude cationic dye from the nucleus during the terminal stage of the apoptotic process. Caspase-3 activity was recorded only in the second polar body. From the morphological point of view, mouse polar bodies acted very similarly to damaged embryonic cells which have lost contact with their neighboring blastomeres. In conclusion, polar bodies possess all the molecular equipment necessary for triggering and executing an active suicide process. Furthermore, similarly as in dying embryonic cells, stressing external conditions (culture in vitro) might accelerate and increase the incidence of apoptotic elimination of the polar bodies in embryos.

Maternal restraint stress negatively influences growth capacity of preimplantation mouse embryos.

In our study we investigated the effect of maternal restraint stress on preimplantation embryo development using a mouse model. We exposed hormonally stimulated (superovulated) and unstimulated (i.e. spontaneously ovulating) mouse females to restraint stress for 30 min three times a day during the preimplantation period. The stress exposure caused significant increase in blood plasma corticosterone concentration. Microscopical evaluation of embryos isolated from spontaneously ovulating females showed that maternal stress significantly increased the proportion of embryos with lower cell numbers (≤32 cells) and decreased the proportion of embryos with higher cell numbers (65-96 cells and 97-128 cells). Moreover maternal restraint stress decreased the cell counts per embryo and per blastocyst. After an additional 24 h in vitro culture we did not find any difference in the embryo distribution or in the cell counts per embryo/blastocyst between embryos isolated from stressed and control mothers. The exposure to restraint stress did not affect the incidence of apoptosis in blastocysts isolated from spontaneously ovulated dams. In gonadotropin stimulated dams, the hormonal treatment itself notably changed embryo distribution (increasing the proportion of degenerated embryos) and increased the occurrence of apoptotic cells. Our results indicate that psychical stress exposure in very early pregnancy can significantly influence the developmental capacity of preimplantation embryos.

Biogenic monoamines in preimplantation development.

BACKGROUND: The involvement of biogenic monoamines in early ('preneural') embryogenesis has been well documented in lower vertebrates, but much less information is available about the role of these molecules in the earliest stages of development in mammals, including humans. METHODS: Databases (PubMed, ISI Web of Knowledge and Scopus) were searched for studies relating to biogenic monoamines functioning in early embryos. The available data on the expression of histamine, serotonin and adrenergic receptors during mammalian preimplantation development were summarized, and the potential roles of biogenic monoamines in very early pregnancy were discussed. RESULTS: The roles of biogenic monoamines in mammalian preimplantation embryo development can be diverse, depending on the embryo developmental stage, and the physiological status of the maternal organism. Several receptors for biogenic monoamines are expressed and biologically functional in cells of preimplantation embryos. Activation of histamine receptors can play a role in embryo implantation and trophoblast invasion. Activation of adrenergic and serotonin receptors can influence proliferation and survival of early embryonic cells. CONCLUSIONS: Biogenic monoamines can play an important role in physiological conditions, contributing to embryo-maternal interactions, or can influence the early embryo under unfavorable or pathological conditions (e.g. in maternal stress, or in women taking certain antidepressants, anti-migraine or anti-ulcer drugs).

Apoptotic cells in mouse blastocysts are eliminated by neighbouring blastomeres.

Apoptosis is a physiological process that occurs commonly during the development of the preimplantation embryo. The present work examines the ability of apoptotic embryonic cells to express a signal promoting their phagocytosis, and quantifies the ability of neighbouring, normal embryonic cells to perform that task. Microscopic analysis of mouse blastocysts revealed phosphatidylserine externalization to be 10 times less common than incidence of apoptotic cells (as detected by TUNEL). In spite of the low frequency of phosphatidylserine-flipping (in inner cell mass, no annexin V staining was recorded), fluorescence staining of the plasma membrane showed more than 20% of apoptotic cells to have been engulfed by neighbouring blastomeres. The mean frequency of apoptotic cells escaping phagocytosis by their extrusion into blastocyst cavities did not exceed 10%. Immunochemically visualised RAC1 (an enzyme important in actin cytoskeleton rearrangement) was seen in phagosome-like structures containing a nucleus with a condensed morphology. Gene transcript analysis showed that the embryonic cells expressed 12 receptors likely involved in phagocytic process (Scarf1, Msr1, Cd36, Itgav, Itgb3, Cd14, Scarb1, Cd44, Stab1, Adgrb1, Cd300lf, Cd93). In conclusion, embryonic cells possess all the necessary mechanisms for recognising, engulfing and digesting apoptotic cells, ensuring the clearance of most dying blastomeres.

Expression of adiponectin receptors and effects of adiponectin isoforms in mouse preimplantation embryos.

BACKGROUND: Adiponectin, a pleiotropic hormone secreted from adipose tissue, can mediate some negative effects of obesity on female health, and can participate in the impaired reproductive performance of obese women. Using a mouse model, we investigated expression of adiponectin receptors in ovulated oocytes and in vivo derived preimplantation embryos, and tested effects of different adiponectin isoforms on development of preimplantation embryos in vitro. METHODS AND RESULTS: Using RT-PCR and immunohistochemistry, we found expression of adiponectin receptors AdipoR1 and AdipoR2, at the mRNA and protein level, in mouse ovulated oocytes and preimplantation embryos. Quantitative real-time RT-PCR analysis showed a decrease in the amount of AdipoR1 and AdipoR2 mRNA after fertilization, which was followed by an increase in mRNA at the morula and blastocyst stage; mRNA for adiponectin was detected only at the blastocyst stage. Administration of full-length adiponectin significantly changed the distribution in numbers of cells of cultured preimplantation embryos, increasing the proportion of embryos with high cell numbers (>128 cells) and decreasing the proportion of embryos with lower cell numbers (<65 cells). Blastocysts possessed significantly higher cell numbers after full-length adiponectin treatment. Mutated trimeric adiponectin had the opposite effect, a significant decrease in the proportion of embryos with higher cell numbers (>96 cells) and increase in the proportion of embryos with lower cell numbers (<65 cells). Trimeric adiponectin also significantly decreased the cell number and increased cell death in blastocysts. Truncated globular adiponectin had no significant effect on development of mouse preimplantation embryos. CONCLUSIONS: Our results indicate that adiponectin can directly influence the development of the preimplantation embryo, and the effects are isoform dependent.

In vitro exposure to pyrethroid-based products disrupts development of mouse preimplantation embryos.

The objective of this study was to evaluate the potential toxicity of pyrethroids (deltamethrin, permethrin, fenvalerate, λ-cyhalothrin), commercial pyrethroid-based products DECIS EW 50 (deltamethrin mixture), TOP SPOT ON STRONGER (permethrin mixture), as well as related secondary ingredients on mouse preimplantation embryo development. Two-cell stage embryos were in vitro cultured with addition of the listed chemicals until blastocyst formation. All active pyrethroids negatively affected embryonic development at 1000 µM concentration. Decreased quality of obtained blastocysts in permethrin, fenvalerate and λ-cyhalothrin-treated embryos was revealed as well. Deltamethrin showed harmful impact on embryo development at 100 µM concentration. Lower concentrations of pyrethroids (1, 10 µM) had no effect on embryo development. The presence of DECIS EW 50 containing deltamethrin at 100 µM caused degeneration of all embryos. Similarly, TOP SPOT ON STRONGER containing 100 µM of permethrin impaired embryonic development and quality of obtained blastocysts. Evaluated secondary ingredients (butylhydroxyanisol, butylhydroxytoluen, butylparaben and cyclohexanone) at corresponding concentrations showed damaging impact on preimplantation embryo development as well. Our results indicate that the embryotoxic potential of active pyrethroids is relatively low, whereas pyrethroid-based products have relatively high potential to impair mouse preimplantation development. Embryotoxicity of commercial products is probably attributable to the presence of secondary ingredients.

A Diabetic Pregnancy Alters the Expression of Stress-Related Receptors in Gastrulating Rabbit Blastocyst and in the Reproductive Tract.

The incidence of diabetes mellitus for young people rises since years. A preconceptional diabetes mellitus leads to subfertility. Most of the causes for a diabetic subfertility are still unknown. Stress can significantly deteriorate glycemic control in diabetes. Several mechanisms by which "stress hormones", like adrenaline and cortisol or corticosterone, can contribute to the regulation of glucose homeostasis have been identified. Using reverse transcription-polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR, we examined the expression of adrenergic receptors and the glucocorticoid receptor transcripts in the female rabbit reproductive tract and in gastrulating blastocysts developed in normoinsulinemic mothers and in mothers with experimentally induced diabetes mellitus type 1. The glucocorticoid receptor expression was detected in the reproductive tract as well as in gastrulating blastocysts at a high level. In maternal endometrium, α1D-, α2A-, ß1-, and ß2-adrenergic receptors were expressed, whereby ß1 transcript was not detectable in the endometrium from diabetic mothers. In preimplantation embryos, all 9 adrenergic receptors were expressed, most of them predominantly in the embryoblast. A maternal diabetes mellitus altered α2A-adrenergic receptor expression in the blastocyst and reversed the ratio of α2A transcript quantity between embryoblast and trophoblast. Our results show that the maternal reproductive tract and the preimplantation embryo express a distinct pattern of the stress response system. Alterations in the pattern and/or in functionality are likely linked to subfertility in diabetes mellitus.

Fipronil causes toxicity in mouse preimplantation embryos.

In this study the possible toxicity of phenylpyrazole fipronil, the related commercial product FIPRON spot-on as well as FIPRON spot-on secondary ingredients on the developmental capacities and quality of mouse preimplantation embryos was evaluated. During in vitro tests, isolated two-cell stage embryos were cultured in media with addition of the listed chemicals until blastocyst formation. Stereomicroscopic evaluation of in vitro produced embryos showed that fipronil at 1 µM and higher concentration negatively affected embryonic development. Fluorescence staining revealed that the obtained blastocysts displayed lower numbers of blastomeres at 10 µM concentrations and elevated incidence of cell death from 1 µM concentration. The presence of FIPRON spot-on at a concentration equivalent to 10 µM of fipronil caused massive degeneration of all embryos. Secondary ingredients (butylhydroxyanisolum, butylhydroxytoluenum) at corresponding concentrations negatively impacted the development and quality of preimplantation embryos as well. During in vivo tests (daily oral administration of fipronil during the preimplantation period) in embryos collected from treated mouse females, significantly elevated incidence of cell death was observed even at the acute reference dose. Fipronil impaired the development and quality of mouse preimplantation embryos in both in vitro and in vivo tests. Embryotoxicity of the commercial product FIPRON spot-on was potentiated by the presence of secondary ingredients.

Relative quantification of mRNA: comparison of methods currently used for real-time PCR data analysis.

BACKGROUND: Fluorescent data obtained from real-time PCR must be processed by some method of data analysis to obtain the relative quantity of target mRNA. The method chosen for data analysis can strongly influence results of the quantification. RESULTS: To compare the performance of six techniques which are currently used for analysing fluorescent data in real-time PCR relative quantification, we quantified four cytokine transcripts (IL-1beta, IL-6 TNF-alpha, and GM-CSF) in an in vivo model of colonic inflammation. Accuracy of the methods was tested by quantification on samples with known relative amounts of target mRNAs. Reproducibility of the methods was estimated by the determination of the intra-assay and inter-assay variability. Cytokine expression normalized to the expression of three reference genes (ACTB, HPRT, SDHA) was then determined using the six methods for data analysis. The best results were obtained with the relative standard curve method, comparative Ct method and with DART-PCR, LinRegPCR and Liu & Saint exponential methods when average amplification efficiency was used. The use of individual amplification efficiencies in DART-PCR, LinRegPCR and Liu & Saint exponential methods significantly impaired the results. The sigmoid curve-fitting (SCF) method produced medium performance; the results indicate that the use of appropriate type of fluorescence data and in some instances manual selection of the number of amplification cycles included in the analysis is necessary when the SCF method is applied. We also compared amplification efficiencies (E) and found that although the E values determined by different methods of analysis were not identical, all the methods were capable to identify two genes whose E values significantly differed from other genes. CONCLUSION: Our results show that all the tested methods can provide quantitative values reflecting the amounts of measured mRNA in samples, but they differ in their accuracy and reproducibility. Selection of the appropriate method can also depend on the design of a particular experiment. The advantages and disadvantages of the methods in different applications are discussed.

Effects of a combination of thyme and oregano essential oils on TNBS-induced colitis in mice.

We examined the anti-inflammatory effects of the combination of thyme and oregano essential oil dietary administered at three concentrations (0.4% thyme and 0.2% oregano oils; 0.2% thyme and 0.1% oregano oils; 0.1% thyme and 0.05% oregano oils) on mice with TNBS-induced colitis. Treatment of colitic animals with the essential oils decreased the mRNA levels of pro-inflammatory cytokines IL-1beta, IL-6, GM-CSF, and TNFalpha, especially after application of the medium dose. The medium dose of the essential oils significantly lowered the amount of IL-1beta and IL-6 proteins too. Moreover, administration of the medium dose decreased the mortality rate, accelerated the body weight gain recovery, and reduced the macroscopic damage of the colonic tissue. Our results indicate that combined treatment with appropriate concentrations of thyme and oregano essential oils can reduce the production of proinflammatory cytokines, and thereby attenuate TNBS-induced colitis in mice.

The Responses of Mouse Preimplantation Embryos to Leptin In Vitro in a Transgenerational Model for Obesity.

The aim of the present study was to test the hypothesis that leptin can directly mediate the negative effect of maternal obesity on preimplantation embryos. As previously shown, maternal obesity retards early embryonic development in vivo and increases the incidence of apoptosis in blastocysts. When two-cell embryos isolated from control and obese mice were transferred to identical (leptin free) conditions in vitro, no differences in any growth or quality parameters were recorded, including apoptosis incidence in blastocysts. Embryos isolated from control mice responded to transfer to environments with a high concentration of leptin (10 ng/mL) with a significant increase in arrest at the first or subsequent cell cycle. However, the majority of non-arrested embryos developed into blastocysts, showing morphology comparable to those cultured in the leptin-free group. On the other hand, the exposure of embryos isolated from obese mice to high leptin concentration in vitro did not retard their development. Furthermore, these embryos developed into blastocysts, showing a lower incidence of apoptosis. In vivo-developed blastocysts recovered from obese mice showed elevated expression levels of the proapoptotic gene BAX and the insulin-responsive glucose transporter gene SLC2A4. In conclusion, elevated leptin levels have both positive and negative effects on preimplantation embryo development in vitro, a response that likely depends on the body condition of the embryo donor. Moreover, these results suggest that leptin acts as a survival factor rather than an apoptotic inductor in embryonic cells. Since no elevations in the expression of the leptin receptor gene (LEPR) or fat metabolism-associated genes (PLIN2, SLC27A4) were recorded in blastocysts recovered from obese mice, the role of leptin in mediating the effects of obesity on embryos at the peripheral level is likely lower than expected.